Osteoarthritis (OA) pain represents an enormous health burden. Patients describe different types of pain over the course of the disease, including pain with mechanical characteristics, neuropathic elements, and intermittent spontaneous pain. Signs of somatosensory disturbances such as mechanical allodynia, loss of proprioception, and vibratory deficits have been reported. Specialized sensory neurons mediate different sensations, including touch, proprioception, vibration, and different types of noxious stimuli. Therefore, we propose that pain in OA is more than merely a state where nociceptive neurons in the joint are activated, but rather involves widespread changes in sensory neurons, both in specialized subsets of nociceptive neurons and in non-nociceptive A fibers. Our overall goal is to identify these changes in subsets of neurons in the course of progressive OA. We will use a validated murine model, destabilization of the medial meniscus (DMM) that captures the long-term progression of OA. In this model, pain-dependent behaviors change over time, with knee hyperalgesia and mechanical allodynia presenting early on, while symptoms of ongoing pain are not apparent until 8-16 weeks post DMM. This provides a unique opportunity to longitudinally assess neuronal properties. Our strategy will include three different approaches: (1) To define changes in specific subsets of sensory neurons in dorsal root ganglia (DRG) and joints in the course of knee OA - Subsets of sensory neurons in the joint and innervating DRG, L2-L5, will be documented throughout the progression of disease, through retrograde labeling and immunohistochemistry. Specifically, we will describe the number and location of peptidergic and non-peptidergic C-fibers, of non-nociceptive A-fibers, and their expression of markers including CCR2 and Nav1.8. We will validate our findings in a unique human joint tissue bank including patients with meniscal pathology, early and advanced stages of knee OA; (2) To characterize physiological properties of subsets of sensory neurons during initiation and maintenance of OA pain- We will analyse activity of individual subsets of A and C-fibers through electrophysiological recordings on intact DRG, L3/L4, and determine spontaneous activity as well as responses to stimuli including MCP-1, capsaicin, and ATP. We will also employ novel Pirt-GCaMP3 mice to image intracellular Ca2+ in sensory neurons in vivo in response to natural sensory stimuli, including mechanical stimulation applied to the knee; (3) To study the effect of selectively silencing subsets of neurons on different OA pain behaviors - We will test the effect of a selective -opioid receptor on pain-dependent behaviors. We will also employ novel DREADD technology to transiently silence all sensory DRG neurons (using the pan-DRG Advillin promoter) or a subset of nociceptors (using Nav1.8 promoter). The proposal combines neuroanatomical techniques, electrophysiological measurements, in vivo imaging, and state-of-the-art approaches to reversibly silence neurons in order to evaluate changes in populations of sensory neurons that contribute to symptoms in a translational OA model.